Kinetic analysis of phospholipase C from Catharanthus roseus transformed roots using different assays

The properties of phospholipase C (PLC) partially purified from Catharanthu s roseus transformed roots were analyzed using substrate lipids dispersed i n phospholipid vesicles, phospholipid-detergent mixed micelles, and phospho lipid monolayers spread at an air-water interface. Using [P-33]phosphatidyl inositol 4,5-bisphosphate (PIP2) of high specific radioactivity, PLC activi ty was monitored directly by measuring the loss of radioactivity from monol ayers as a result of the release of inositol phosphate and its subsequent d issolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extreme s of initial pressure. The optimum surface pressure for PIP2 hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP2 vesicles followed by ultracentrifugation demonstrated stable attachmen t of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determi ne whether PLC activity was dependent on both bulk PIP2 and PIP2 surface co ncentrations in the micelles. The interfacial Michaelis constant was calcul ated to be 0.0518 mol fraction, and the equilibrium dissociation constant o f PLC for the lipid was 45.5 mu M. These findings will add to our understan ding of the mechanisms of regulation of plant PLC.